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  • Bruno Vellutini 10:54 on 2014/11/27 Permalink
    Tags: , , , , , , , , pou, ,   

    Novocrania and double in situs of Terebratalia 

    Nano ptc1 Nano smo1 Nano smo2 Nano gli
    Nano pax6 Nano delta Nano notch
    Ttra pax6 (DNP/cy5) + pax258 (DIG) Ttra en (DNP) + pax6 (DIG) Ttra wnt1 (DNP) + delta (DIG) Ttra wnt1 (DNP) + pou4 (DIG)


    Added probes at 1 ng/µL concentration.


    Washes, incubated with the blocking for 1h and at 4 °C overnight with the respective antibodies. Anti-DIG-AP 1:5000 for all Novocrania wells and Anti-DIG-POD 1:250 for all Terebratalia wells.


    Washed antibody from all samples with:

    • 5x 15 min PBT and 5x 30 min PTw.
    • 3x 5min washes of AP minus MgCl2 for regular in situ and TNT buffer for fluorescent in situs.

    Developed fluorescent wells with TSA kit using cy3 fluorochrome for 2 hours. Sropped the reaction with 5x 5 min detergent solution at 60°C followed by PTw washes. POD inactivation with 0.1% H2O2 for 45 min, PTw washes, and finally formamide based POD inactivation buffer for 15 min at 60 °C. Signal for pax6 and delta were very strong, pou4 was strong and pax258 was medium.

    I started developing Novocrania with NBT/BCIP. Pax6 was the first to come, strongly. The others were slower, but gli had a good signal/noise ratio. Others acquired background after the first AP change after 2 hours. I stopped all, except delta and smo2 (which had less background).


    Today they were quite dark :/

    Went through ethanol washes and antibody washes for fluorescent. Added 70% glycerol in PTw with 1:10000 sytox green.

  • Bruno Vellutini 15:31 on 2014/11/25 Permalink
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    Cloning Novocrania patched and longer clones of wnt1, wnt5 

    I made new primers for ptc2 and longer clones for wnt1 and wnt5. Set PCR overnight.


    Ran gel and cut a band, but wnt1 and ptc2 did not work… OF COURSE! because fragment lengths are the following: wnt1=1239 bp, wnt5=1097 bp, ptc2=1258 bp! And the elongation time of the PCR was set to X.

    2014-11-26 11.16.21


    Re-tried the same PCR with a longer elongation time. The result was the same. Chema suggested running nested primers to see if something appears.

    2014-11-28 17.18.15

  • Bruno Vellutini 15:29 on 2014/11/25 Permalink
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    Probe synthesis Novocrania delta/notch 

    Probe PCR set for Novocrania delta (BV348: Sp6) and notch (BV 350: Sp6).

    2014-11-25 16.44.03

    Cut and extracted.


    Set transcription reaction for 7h. Added DNAse and 5 µL of lithium.


    Finishing up.

    ng /µL
    Nano delta 1978
    Nano notch 1571
    Tt pou4 371


  • Bruno Vellutini 18:48 on 2014/11/22 Permalink
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    Terebratalia segmentation in situ 

    In order to have more temporal resolution of engrailed I am adding one well per stage. In addition some segmentation related genes.

    en cleavage+blastula en radial gastrula en asym gastrula en bilateral gastrula en trilobed+early+late larva
    pax6 nk1a nk1b lmx1
     lfng runx hh


    Washed probe out. Added antiDIG-AP antibody at 1700 and incubated overnight at 4 °C.


    Washed antibody and started developing. Pax6 came up quite fast. I exchanged the solution after 1h and stopped after 2h. The remaining are slowly coming up, except asym and bilateral of engrailed. I used one tube of 0.8 ng/µL of probe and it must have been even lower concentration because the other 3 engrailed wells are coming up ok (same probe batch, but from another tube). Another issue is that there is not early gastrula like I wanted, they seem to be already have the differentiated lateral patches.

    Lunatic fringe and runx are not showing up. I exchanged AP from all wells except engrailed after 3h and then all wells before leaving at 4 °C overnight.


    Same as yesterday with NK1s and lmx coming up nicely. Exchanged AP at 10:30.


    Kept developing for 7h and stopped all wells. Nothing came up for lfng, runx or hedgehog.


    Ethanol washes for all. Added 70% glycerol with 1:10000 sytox green in hedgehog well. I want to see if embryos turn red… If not I’ll check if it is better than DAPI for these regular in situs.

  • Bruno Vellutini 19:31 on 2014/11/18 Permalink
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    Probe synthesis of Novocrania Hedgehog pathway 

    Selected minipreps for probe synthesis of Novocrania Hedgehog pathway and remaining Terebratalia genes. From this post.

    BV326 Terebratalia transversa Lmx1 T7 ok + SP6
    BV328 Terebratalia transversa NK1a T7 ok + SP6
    BV330 Terebratalia transversa NK1b T7 ok + SP6
    BV332 Novocrania anomala Patched1 T7 ok + SP6
    BV336 Novocrania anomala Smoothened1 T7 ok + SP6
    BV338 Novocrania anomala Smoothened2 T7 ok + SP6
    BV340 Novocrania anomala Gli T7 ok + SP6
    BV343 Novocrania anomala Pax6 T7 ok + SP6
    BV345 Novocrania anomala Pax3/7 T7 ok T7
    BV346 Novocrania anomala NK1 T7 ok + SP6
    TTR Terebratalia Pax6 T7 DIG
    TTR Terebratalia Pax6 T7 DNP

    Set probe PCR to run overnight.


    Ran gel and painfully extracted the PCR…

    2014-11-19 19.09.46


    Started probe synthesis for the genes above at 9:30. Finished at 16:30 and precipitated overnight.


    gene ng/µL goal concentration volume total volume volume of hybe buffer
    Ttra lmx1 906 50 24 426 402
    Ttra nk1a 974 50 24 458 434
    Ttra nk1b 1098 50 24 516 492
    Nano ptc1 1075 50 24 505 482
    Nano smo1 2041 50 24 959 936
    Nano smo2 1830 50 24 860 836
    Nano gli 488 50 24 229 206
    Nano pax6 1350 50 24 634 611
    Nano pax37 2224 50 24 1045 1022
    Nano nk1 1050 50 24 494 470
    Ttra pax6 dig 1076 50 24 506 482
    Ttra pax6 dnp 50 50 24 23 0


  • Bruno Vellutini 19:26 on 2014/11/18 Permalink
    Tags: , , , , , , ovo, , ,   

    Transformation for remaining Terebratalia and Novocrania 

    Terebratalia: ovo, serrate, pygopus, legless (from here)

    Novocrania: notch, delta, gbx (from here)

    Transformed and plated with 400 µL.


    Colony PCR.

    2014-11-19 19.09.53

    Set Nano delta and notch to grow.


    Extracted minipreps for Nano notch and delta:

    gene id seq primer antisense
    Nano delta BV348 T7 SP6
    Nano delta BV349 T7 SP6
    Nano notch BV350 T7 SP6
    Nano notch BV351 T7 T7

    Set sequencing PCR for them.


    Collected sequences. See orientation above.

  • Bruno Vellutini 18:49 on 2014/11/11 Permalink
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    Transformation to sequencing of Novocrania 

    I selected the 8 most important Novocrania genes from the first and second batched of cloning: ptc1, ptc2, smo1, smo2, gli, pax6, pax3/7, nk1

    Transformed and plated.


    Set colony PCR:

    2014-11-12 15.43.52


    BV332 Novocrania anomala Patched1 T7 ok + SP6
    BV333 Novocrania anomala Patched1 T7 ok + SP6
    BV334 Novocrania anomala Patched2 T7 ?
    BV335 Novocrania anomala Patched2 T7 ?
    BV336 Novocrania anomala Smoothened1 T7 ok + SP6
    BV337 Novocrania anomala Smoothened1 T7 ok + SP6
    BV338 Novocrania anomala Smoothened2 T7 ok + SP6
    BV339 Novocrania anomala Smoothened2 T7 ok + SP6
    BV340 Novocrania anomala Gli T7 ok + SP6
    BV341 Novocrania anomala Gli T7 ok T7
    BV342 Novocrania anomala Pax6 T7 ok T7
    BV343 Novocrania anomala Pax6 T7 ok + SP6
    BV344 Novocrania anomala Pax3/7 T7 ok T7
    BV345 Novocrania anomala Pax3/7 T7 ok T7
    BV346 Novocrania anomala NK1 T7 ok + SP6
    BV347 Novocrania anomala NK1 T7 ok + SP6


  • Bruno Vellutini 16:15 on 2014/11/10 Permalink
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    Cloning Hedgehog components of Novocrania 

    PCR with diluted primers for 40 cycles instead of 34 for: ptc1, ptc2, smo1, smo2, gli

    2014-11-10 22.32.27

    Had two bands for some. Set a ligation overnight for all at 4 °C.

  • Bruno Vellutini 18:33 on 2014/11/07 Permalink
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    Novocrania cloning 

    Set PCR for gbx, pax6, pax3/7, nk1, notch, delta.


    Ran gel and set ligation.

    2014-11-08 15.29.44

  • Bruno Vellutini 19:19 on 2014/11/06 Permalink
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    Testing new Novocrania cDNA 

    I’m testing the new cDNA for Novocrania made by Daniel. He made a mix of extractions from different stages and diluted 1:10 and 1:20. I tested with previous genes that worked, pax2/5/8 and wnt11. Set the PCR with a reaction for the 1x, 1:10 and 1:20.


    2014-11-07 12.17.42

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